Reassessing Sex Differences: Behavioral Analysis of Male and Female Wistar Rats in the Open Field Test

This study aimed to investigate sex-specific behavioral differences in adult Wistar rats using a 3-minute Open Field Test (OFT), aligning with ethical guidelines emphasizing inclusive animal research for translational validity. While previous literature suggests female Wistar rats often display greater locomotor activity and central exploration, indicative of lower anxiety, these findings are not universal due to protocol variations. Fourteen Wistar rats (7 males, 7 females), aged 12 weeks, underwent the OFT in a controlled environment, adhering strictly to ethical protocols. Behavioral parameters assessed included locomotor activity, time in central zone, rearing frequency, grooming, and defecation. Data were analyzed using Student's t-test or Mann-Whitney test, with a significance level of p < 0.05. Results revealed no statistically significant differences between sexes for any analyzed variable (p > 0.05). Females exhibited numerically higher locomotor activity (50.43 ± 10.0 vs. 45.29 ± 12.3) and rearing frequency (17.43 ± 5.56 vs. 14.29 ± 4.15), whereas males showed numerically higher grooming (2.43 ± 2.51 vs. 0.14 ± 0.38) and defecation (1.71 ± 1.5 vs. 0.43 ± 1.13). These numerical trends, however, did not reach statistical significance. The findings align with the mixed results reported in the literature concerning sex differences in anxiety and exploratory behaviors within the OFT paradigm, particularly when using small sample sizes. Methodological limitations include the sample size and the absence of estrous cycle control, though the latter was reinterpreted for its ecological validity. Despite the lack of robust differences in spatial or general activity metrics, the observed trends in grooming and defecation hint at subtle sex-specific stress reactivity. This study contributes to methodological optimization by demonstrating the applicability of a brief 3-minute OFT protocol, which reduces animal exposure to experimental stress. Furthermore, it reinforces the critical importance of systematically including both sexes in behavioral research to ensure translational validity, even when statistically robust differences are not immediately apparent. Future research incorporating larger sample sizes and comprehensive hormonal monitoring is necessary for a more nuanced characterization of sex-specific behavioral responses in stress-related paradigms.